CN110903991A

CN110903991A - Recombinant pichia pastoris engineering bacteria containing high-copy-number humanized lysozyme gene and application thereof

Info

Publication number: CN110903991A
Application number: CN201911103534.6A
Authority: CN
Inventors: 陈小龙; 周斌; 朱林江; 陈艺强; 陆跃乐; 朱勇刚; 陈翰驰
Original assignee: ZHEJIANG SILVER-ELEPHANT BIO-ENGINEERING Co Ltd; Zhejiang University of Technology ZJUT
Current assignee: ZHEJIANG SILVER-ELEPHANT BIO-ENGINEERING Co Ltd; Zhejiang University of Technology ZJUT
Priority date: 2019-11-13
Filing date: 2019-11-13
Publication date: 2020-03-24

Abstract

The invention discloses a recombinant pichia pastoris engineering strain containing a high-copy-number humanized lysozyme gene and application thereof, wherein the recombinant pichia pastoris engineering strain is obtained by integrating a lysozyme gene shown as SEQ ID NO.1 into a pichia pastoris genome at a high copy number. The invention adopts two plasmid-mediated recombinant bacterium construction methods to obtain a recombinant bacterium with the copy number of 15; the recombinant bacterium has high lysozyme production activity, and the fermentation enzyme activity is 180-220KU/mL within 120-140 h.

Description

Recombinant pichia pastoris engineering bacteria containing high-copy-number humanized lysozyme gene and application thereof

(I) technical field

The invention relates to recombinant pichia pastoris containing high-copy-number humanized lysozyme genes and application thereof in fermentation production of humanized lysozyme.

(II) background of the invention

Lysozyme is an effective antibacterial agent which specifically acts on cell walls, β -1, 4-glycosidic bond between N-acetylglucosamine which destroys cell walls and N-acetylmuramic acid decomposes insoluble mucopolysaccharide into soluble glycopeptide, and has bacteriolytic action, the lysozyme has wide sources in nature and can be divided into four types of lysozyme including microorganisms, bacteriophage, plants and animals, wherein the lysozyme has high activity and wide application, FAO/WTO in 1992 discloses that the lysozyme is safe when applied to food industry, and the lysozyme is approved by No. 23 bulletin in No. 2010 of China Ministry of public health as food additive.

The lysozyme is a basic globulin, the isoelectric point is 10.7-11.3, the relative molecular weight is 14.3kDa, the optimum pH is 6-7, the optimum temperature is 50 ℃, and 4 disulfide bonds are arranged in the molecule. At present, lysozyme is mainly extracted from egg white and eggshell membrane. However, the production method of direct extraction has small scale and high cost, and is not suitable for large-scale production and application. In particular, as the management of antibiotics for feed is increasingly enhanced, the market demand for lysozyme is rapidly expanding. The lysozyme Escherichia coli expression system and the yeast expression system are constructed by utilizing the genetic engineering technology, so that the lysozyme can be subjected to heterologous expression and microbial fermentation production, and the method becomes a hotspot of the research on the lysozyme production technology. At present, a lot of patents are applied in construction of lysozyme engineering bacteria, including egg white lysozyme (CN101050467B, CN104263709A, CN104694559A, CN105039189B and CN104946675A), human lysozyme (CN1325958, CN1300852A, CN1289678C, CN1664096A, CN102229939B, CN104278017A, CN109295033A and CN109295088A), swine lysozyme (CN107236681A, CN105861468A and CN106046173A) and the like. In order to improve the expression capacity of lysozyme, the invention integrates the expression reading frame of artificially assembled multicopy humanized lysozyme gene into a pichia pastoris genome through two integrative plasmids, selects multicopy integrative engineering strains and constructs the pichia pastoris engineering strain with high copy number integration of the humanized lysozyme.

Disclosure of the invention

The invention aims to provide a pichia pastoris engineering bacterium containing a high copy number humanized lysozyme gene, and the method realizes the efficient production of the humanized lysozyme by a fermentation method.

The technical scheme adopted by the invention is as follows:

in a first aspect, the invention provides a recombinant pichia pastoris engineering strain containing a high-copy-number humanized lysozyme gene, which is obtained by integrating a pichia pastoris genome with a high copy number, wherein the pichia pastoris engineering strain is derived from a Homo sapiens humanized lysozyme gene shown in SEQ ID NO.1 and is subjected to codon optimization. The Pichia pastoris is preferably Pichia pastoris (Pichia pastoris) GS115, and the Pichia pastoris engineering strain is recorded as recombinant Pichia pastoris (Pichia pastoris) IEF-hlyz 15.

Further, the high copy number means that the copy number of the egg white lysozyme gene is 15.

Furthermore, the amino acid sequence of the human lysozyme gene coding Protein is SEQ ID NO.2, and the NCBI database accession number GenBank Protein ID: p61626.

The construction method of the recombinant Pichia pastoris engineering bacteria (preferably Pichia pastoris IEF-hlyz15) comprises the following steps:

1) synthesizing a codon-optimized human lysozyme gene with a nucleotide sequence SEQ ID NO.1 by a gene synthesis company, and cloning the codon-optimized human lysozyme gene to a secretory expression plasmid pPIC9K to obtain a recombinant plasmid pPIC9K-hlyz shown in figure 1;

2) after the pPIC9K-hlyz is linearized by using a restriction endonuclease SacI, the strain is transformed into a Pichia pastoris GS115 strain, high-concentration G418 resistant plate screening, transparent ring plate high-throughput screening and triangular flask fermentation experiments are carried out, a high-copy seed and high-activity recombinant strain HC243 are obtained through screening, and quantitative PCR analysis shows that the copy number of the humanized lysozyme gene is 5. The high-concentration G418 resistant plate is a YPD plate of 3.5G/L geneticin G418;

3) cloning the expression reading frame of the human lysozyme gene on pPIC9K-hlyz to pPICZ α A plasmid, and constructing a recombinant expression plasmid pPICZ α -hlyzV containing 5 copy numbers, as shown in FIG. 2;

4) the method comprises the steps of linearizing pPICZ α -hlyzV by using restriction enzyme SalI, converting the recombinant strain HC243 obtained by screening, screening by a Zeocin resistance plate, a transparent ring plate high-throughput screening and a triangular flask fermentation experiment to obtain a high copy number and a high-activity recombinant strain IEF-hlyz15, and marking the strain as recombinant Pichia pastoris (Pichia pastoris) IEF-hlyz15, wherein the copy number of a human lysozyme gene is 15 through quantitative PCR analysis.

In a second aspect, the invention provides an application of the recombinant pichia pastoris engineering bacteria (preferably strain pichiapastoris ief-hlyz15) in preparing lysozyme through fermentation.

The fermentation method comprises the following steps: (1) streaking the recombinant Pichia engineering bacteria (preferably Pichia pastoris IEF-hlyz15) preserved in glycerol tube onto YPD plate, and culturing at 25-30 deg.C (preferably 30 deg.C) for 40 hr; inoculating the single colony into a seed culture medium, culturing at 25-30 ℃ and 100-250rpm (preferably 30 ℃, 200rpm and 20h) to the middle logarithmic growth phase to obtain a seed solution; the YPD plates consisted of: 10g/L of yeast powder, 20g/L of peptone, 10g/L of glucose and 15g/L of agar powder, wherein the solvent is deionized water, and the pH value is natural; the final concentration composition of the seed culture medium is as follows: 10g/L of yeast powder, 20g/L of peptone and 10g/L of glucose, wherein the solvent is deionized water and the pH value is 7.0;

2) fermentation culture: inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 1-10% (preferably 4%) by volume for glycerol batch fermentation at 25-30 ℃, wherein the dissolved oxygen DO is greater than 20%, the glycerol is exhausted after fermentation for 18-24h, and the dissolved oxygen DO value is rapidly increased; starting to enter a glycerol feeding fermentation stage: the glycerol feed supplement culture medium is supplemented at a rate of 18-20mL/h/L of initial fermentation broth volume, the fermentation is carried out for 4-6h (preferably at a temperature of 30 ℃ and an air flow of 4L/min/L) under the conditions of 25-30 ℃, a stirring rate of 200-900 rpm and an air flow of 2-4L/min/L of initial fermentation culture medium, the stirring rotation speed is controlled to be coupled with a dissolved oxygen DO value in the fermentation process, the DO value is controlled to be more than 20%, the glycerol feed supplement culture medium is 15-20% of the volume of the original added fermentation culture medium, glycerol is exhausted, dissolved oxygen is rapidly increased, and starvation culture is continued for 1-2 h; entering a methanol induction fermentation stage, controlling the feed rate of a methanol feed medium to be 3.0-3.6mL/h/L of an initial fermentation medium, maintaining the dissolved oxygen DO to be more than 10%, fermenting for 2-3h, increasing the feed rate of the methanol feed medium to be 5.0-7.2mL/h/L of the initial fermentation medium, after fermenting for 3-5h, continuously increasing the feed rate of the methanol feed medium to be 9.0-10.2mL/h/L of the initial fermentation medium, continuously fermenting for 110-120h, and finishing fermentation to obtain fermentation liquor containing lysozyme; the supplementing amount of the methanol supplementing culture medium is 50-80% of the volume of the fermentation culture medium which is originally added; the fermentation medium comprises the following components in final mass concentration: 26.7 ml/L85% phosphoric acid, 0.93g/L calcium sulfate, 18.2g/L potassium sulfate, 14.9g/L MgSO₄·7H₂O, 4.13g/L of potassium hydroxide, 40g/L of glycerol, 4.35ml/L of PTM1 buffer solution, deionized water as a solvent and pH5.0; the glycerol feed medium comprises the following components: 500mL/L of glycerol with the mass concentration of 25 percent and 12mL/L of PTM1 buffer solution; the methanol feed medium comprises the following components: 12ml/L PTM1 buffer solution and anhydrous methanol; the PTM1 buffer composition: 6.0g/L CuSO₄·5H₂O, 0.08g/L sodium iodide, 3.0g/L MnSO₄·H₂O，0.2g/L Na₂MoO₄·2H₂O, 0.02g/L boric acid, 0.5g/L cobalt chloride, 20.0g/L zinc chloride and 65.0g/L FeSO₄·7H₂O, 0.2g/L biotin, 5.0 ml-L sulfuric acid, and the solvent is deionized water.

Compared with the prior art, the invention has the beneficial effects that ① adopts two plasmid-mediated recombinant bacterium construction methods to obtain the recombinant bacterium with high copy number, the copy number of the genome of the human lysozyme is 15, the ② recombinant bacterium has high lysozyme production activity, and the lysozyme activity is 180-fold and 220KU/mL within 120-fold and 140h, and is higher than the fermentation level (170 KU/mL) reported in the prior patent CN 107794274A (application No. 201711022926.0).

The invention relates to a Pichia pastoris IEF-hlyz15 of recombinant Pichia pastoris engineering bacteria for producing human lysozyme by fermentation, which is directly fermented to produce the human lysozyme. The pichia pastoris has the characteristic of food safety, no antibiotic is added in the fermentation process, and the humanized lysozyme prepared by fermentation is suitable for the food and feed industries.

(IV) description of the drawings

FIG. 1 shows the spectrum of the recombinant plasmid pPIC9 k-hlyz.

FIG. 2 is a map of recombinant plasmid pPICZ α -hlyzV.

FIG. 3 is a graph showing the results of plate screening based on the lysis ring.

FIG. 4 is a map of a standard plasmid pUC18-GAPDH for copy number detection.

FIG. 5 is a standard curve of copy number of GAPDH gene.

FIG. 6 is a standard curve of copy number of hlyz gene of human lysozyme.

FIG. 7 is a graph of the variation of high density fermentation parameters of the strain Pichia pastoris IEF-hlyz 15.

FIG. 8 is a graph showing the variation of cell density OD value and lysozyme activity of high-density fermentation of the strain Pichia pastoris IEF-hlyz 15.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

in the examples of the present invention, unless otherwise specified, all methods used are conventional ones, and all reagents used are commercially available.

LB culture medium: 5.0g/L yeast powder, 10g/L peptone, 10g/L NaCl and deionized water as solvent, and the pH value is 7.0.

Low-salt LB medium: 5.0g/L yeast powder, 10g/L peptone, 5g/L NaCl and deionized water as solvent, and the pH value is 7.0.

YPD medium: 10.0g/L yeast powder, 20g/L peptone, 20g/L glucose and deionized water as solvent, and the pH is natural.

MD culture medium: 1.34g/L yeast nitrogen source alkali (YNB), 0.4mg/L biotin, 10g/L glucose, deionized water as solvent, and natural pH.

MM solid medium composition: 1.34g/L yeast nitrogen source alkali (YNB), 0.4mg/L biotin, 5mL/L methanol, 15g/L agar powder, 100mM pH6.0 potassium phosphate buffer.

BMGY medium: l0g/L yeast powder, 20g/L peptone, 1.34g/L YNB, 0.4mg/L biotin, 10g/L glycerol, 100mM potassium phosphate buffer, pH 6.0.

BMMY medium: yeast powder L0g/L, peptone 20g/L, 1.34g/L YNB, 0.4mg/L biotin, 5mL/L methanol, 100mM pH6.0 potassium phosphate buffer.

The final concentration of the fermentation medium is as follows: 26.7 ml/L85% phosphoric acid, 0.93g/L calcium sulfate, 18.2g/L potassium sulfate, 14.9g/L MgSO₄·7H₂O, 4.13g/L of potassium hydroxide, 40g/L of glycerol, 4.35ml/L of PTM1 buffer solution, deionized water as a solvent and pH 5.0.

The glycerol feed medium was prepared by adding 12ml/L of PTM1 buffer to 25% by mass of glycerol.

The methanol feed medium was prepared by adding 12ml/L of PTM1 buffer to anhydrous methanol.

PTM1 buffer: 6.0g/L CuSO₄·5H₂O, 0.08g/L sodium iodide, 3.0g/L MnSO₄·H₂O，0.2g/LNa₂MoO₄·2H₂O, 0.02g/L boric acid, 0.5g/L cobalt chloride, 20.0g/L zinc chloride and 65.0g/L FeSO₄·7H₂O, 0.2g/L biotin, 5.0ml/L sulfuric acid and deionized water as solvent, filtering and sterilizing.

Example 1 Artificial Synthesis of human lysozyme Gene hlyz derived from Homo sapiens and construction of pPIC9K-hlyz plasmid

According to the amino acid sequence (SEQ ID NO.2) of human lysozyme from Homo sapiens with NCBI database GenBank Protein ID P61626, the nucleotide sequence (SEQ ID NO.1) is obtained by optimizing according to the codon preference of pichia pastoris, the gene synthesis is carried out by Wuxi Huada Qinglan biotechnology limited, the gene is cloned between EcoRI and NotI of pPIC9K, a recombinant plasmid pPIC9K-hlyz is obtained, as shown in figure 1, the recombinant plasmid is transformed into an E.coli DH5 α strain, and the E.coli DH5 α (pPIC9K-hlyz) recombinant strain is obtained.

Example 2 construction of secretory expression of recombinant Pichia pastoris for human lysozyme

After the linearized pPIC9K-hlyz plasmid is transformed into Pichia pastoris (Pichia pastoris) GS115, resistant strains are screened by a high-concentration G418 plate, strains with high-yield human lysozyme are screened by a lysis ring plate, and finally, a recombinant Pichia pastoris strain secreting and expressing human lysozyme is obtained by a triangular flask shaking experiment, wherein the specific embodiment is as follows:

① linearizing pPIC9K-hlyz plasmid, inoculating strain E.coli DH5 α (pPIC9K-hlyz) to LB culture medium containing 100 mug/mL ampicillin, culturing overnight at 37 ℃, extracting pPIC9K-hlyz plasmid by using a plasmid extraction kit, digesting the plasmid pPIC9K-hlyz by using restriction enzyme SacI, linearizing the plasmid, performing gel recovery on the digested product, and dissolving the purified linearized plasmid in ultrapure water;

② making competent cells of Pichia pastoris GS115, inoculating single colony of Pichia pastoris GS115 into 250mL triangular flask containing 25mL LYPD liquid culture medium, culturing overnight at 30 deg.C, transferring into 500 triangular flask containing 50mL YPD liquid culture medium at 1% volume concentration, and culturing overnight at 30 deg.C until OD₆₀₀1.5; 1500 Xg, 4 ℃ under the conditions of centrifugal culture solution for 5min, with 50mL ice bath ultrapure water heavy suspension cell; 1500 Xg, 4 ℃ under the conditions of centrifugal culture solution for 5min, then with 25mL ice bath ultrapure water heavy suspension cell; 1500 Xg, centrifuging the culture solution at 4 deg.C for 5min, and resuspending the cells with 2mL of 1M sorbitol aqueous solution; the culture solution is centrifuged at 1500 Xg and 4 ℃ for 5min, and finally the cells are resuspended in 100. mu.L ice-bath 1M sorbitol aqueous solution to make the bacterial suspension volume about 150. mu.L, and the competent cells of Pichia pastoris GS115 are obtained.

③ transformation of Pichia pastoris GS115, selection of positive transformants, adding 80 μ L of ② competent cells of Pichia pastoris GS115 and 10 μ g of plasmid pPIC9K-hlyz linearized in the step ① into a 1.5mL pre-cooled centrifuge tube, mixing to obtain transformation mixture, transferring the transformation mixture into a transformation cup (0.2cm type) pre-iced with ice, ice-cooling for 5min, performing electric transformation by using a Bio-Rad electric shock instrument with voltage of 1.5kV, electric field intensity of 7.5kV/cm, capacitance of 25 μ F and resistance of 400 Ω, immediately adding 1mL of 1M sorbitol aqueous solution in ice-bath into the transformation cup after pulse, transferring the transformation solution into a new 1.5mL centrifuge tube, standing at 30 ℃ for 2h, taking 200 μ L of culture solution, coating an MD plate, and culturing at 30 ℃ until transformants appear.

④ screening colonies resistant to high concentration G418, scraping the colonies on MD plate, diluting with sterile water in 10 times gradient, spreading 100 μ L bacterial suspension with different dilution gradient on YPD plate containing 3.5G/L geneticin G418, and culturing at 30 deg.C for 48 hr until colonies appear.

⑤ screening recombinant strains of human lysozyme by lysis ring plate method, inoculating Micrococcus murauliticus ATCC4698 in 50mL LB liquid culture medium, culturing at 37 deg.C for 18h, centrifuging at 3000 Xg for 10min, discarding supernatant, collecting cells, resuspending the cells in 5mL sterile water, adding to 100mL BMMY solid culture medium heated, boiled and melted, mixing Micrococcus cells with the culture medium to make into plate with Micrococcus, dropping the colony growing on YPD plate containing G418 in step ④ onto the plate with Micrococcus, culturing at 30 deg.C for 3-5 days as shown in FIG. 3, and observing change of size of lysis ring continuously.

⑥ triangular flask shake flask fermentation for screening high-yield strain of anthropogenic lysozyme the shake flask fermentation experiment is divided into two stages, namely cell growth stage in BMGY culture medium and induced enzyme production stage in BMMY, single colony is picked up and inoculated into 5mL BMGY culture medium in 50mL centrifuge tube, shake flask culture is carried out at 30 ℃ for 18h to fermentation liquor OD₆₀₀When the concentration is 2.0, taking out the centrifugal tube, centrifuging at 2000 Xg for 10min, and pouring out the supernatant; resuspending the cells in 5mL BMMY medium, addThe cells were put into a 250mL Erlenmeyer flask containing 20mL of BMMY medium, and a total of 25mL of BMMY medium was subjected to induction culture at 30 ℃ in a shaker at 200 rpm. After every 24h, 200 mu L of fermentation liquor is taken out, and 200 mu L of anhydrous methanol is supplemented at the same time, and the continuous induction fermentation is carried out for 3 d. And taking out a fermentation liquor sample for lysozyme enzyme activity determination.

The lysozyme activity determination method refers to the detection method of the national food safety standard GB 1886.257-2016.

Definition of enzyme activity unit: one unit of enzyme activity (U) is defined as the amount of lysozyme required to cause a change in absorbance at 450nm per minute to 0.001 in a suspension of Micrococcus muralis at 25 ℃ and pH 6.2.

Preparation of a suspension of Micrococcus paridis 0.5g of ⑤ cells of Micrococcus paridis ATCC4698 containing 0.372g/LEDTANa₂50ml of a cell suspension of the muramidase was prepared in 0.1mol/L sodium phosphate buffer solution (pH6.2), and the cell suspension was cultured in a constant temperature shaker at 28 ℃ for 30min before use. The suspension is stable at room temperature for 2h to contain 0.372g/L EDTANA₂The pH of the buffer solution is 6.2, the zero point of a spectrophotometer is adjusted, and the light absorption value of the wall-dissolving microballon bacterial suspension is measured, wherein the reading at 450nm is about 0.70 +/-0.1.

Placing a 1cm cuvette into a spectrophotometer at room temperature at 25 deg.C and using a solution containing 0.372g/L EDTANA₂The absorbance zero point was adjusted with the phosphate buffer (pH6.2). And (3) sucking 2.9mL of the wall-dissolving microballon bacterial suspension into a cuvette, wherein the absorbance at the initial 450nm position should be 0.70 +/-0.10, and the measurement can be started when the initial absorbance change within 3min is less than or equal to 0.003. Sucking 0.1mL of fermentation liquid sample to be detected, adding the wall-dissolving microspherical bacteria suspension, and fully mixing to prepare a sample solution. The change in absorbance at 3min was recorded, and the absorbance was recorded every 15 s. The change of absorbance value per minute should be 0.03-0.08, and the concentration of sample solution should be adjusted if it is not in the required range. The operation was repeated to measure the sample solution. The reaction was stable after 1min and the initial 1min reading was ignored for the calculations.

The above-mentioned calculation of the enzyme activity X result was carried out using the following formula (1):

X＝((A1-A2))/(2×v×0.001) (1)

in the formula:

a1-absorbance of the sample solution at 450nm when reacted for 1 min;

a2-absorbance of the sample solution at 450nm when reacted for 3 min;

v-volume of sample in sample solution for analysis in milliliters (mL)

2-time taken to obtain absorbance readings of 1min and 3min in minutes (min)

0.001-value of the decrease in absorbance caused by the unit lysozyme per minute.

Through the triangular flask fermentation experiment, a strain with high egg white lysozyme yield is obtained through screening and is marked as a recombinant strain HC 243.

The copy number of the human lysozyme gene in the strain HC243 is detected by adopting a double-standard curve method of fluorescent quantitative PCR:

① construction of a Standard plasmid pUC18-GAPDH for the reference gene GAPDH (glyceraldehyde-3-phosphate-dehydrogenase gene), as shown in FIG. 4 genomic DNA of Pichia pastoris GS115 strain was extracted using the following primer Gap-pUC18-F:

CGAATTCGAGCTCGGTACCCGGGGATCCTTTTTTGTAGAAATGTC and Gap-pUC 18-R: CGACGGCCAGTGCCAAGCTTTTTAGATAAGGACGGAGAGATG, amplifying GAPDH, extracting pUC18 plasmid, carrying out double digestion by BamHI/HindIII, recovering and purifying gel, carrying out ligation reaction on the amplified and purified GAPDH and the purified linearized plasmid pUC18 plasmid by a one-step cloning kit, transforming E.coli DH5 α strain, screening by an ampicillin resistance plate to obtain positive clone, and obtaining the standard plasmid pUC18-GAPDH by sequencing verification, wherein the standard plasmid is shown in figure 4.

② preparation of RT-PCR double standard curve:

detecting the mass concentration of pUC18-GAPDH plasmid and pPIC9k-hlyz plasmid by using a trace nucleic acid quantifier; the copy number is scaled according to the following equation: copy number 6.023 × 1014 × plasmid mass concentration/(660 × M), where M denotes the length of the gene (bp) tested. The plasmid solution was diluted to 10 with double distilled water⁴、10⁵、10⁶、10⁷And 10⁸Plasmid gradient dilutions of each copy/. mu.L, separatelymu.L of each plasmid gradient dilution was used as a template, and RT-gap-F (TTGTCGGTGTCAACGAGGAGGAG)/RT-gap-R (GGTCTTTTGAGTGGGCGGTC), RT-hlyz-F (GGCTAACTGGATGTGTTTGGC)/RT-hlyz-R (AAGCGTTAACAGCACCTGGA) were used as primers to perform fluorescent quantitative PCR analysis. Each sample was tested in duplicate 3 times, with 3 replicates for each test. The Ct value given by the fluorescent quantitative PCR was used as the ordinate and the plasmid copy number was used as the abscissa to establish a dual-standard curve, as shown in FIGS. 5 and 6.

③ determination of copy number of human lysozyme gene in recombinant strain HC243

And (3) performing fluorescence quantitative PCR by taking 1 mu L of recombinant strain HC243 genome DNA as a template and respectively taking RT-gap-F/RT-gap-R and RT-hlyz-F/RT-hlyz-R as primers, and substituting the obtained Ct values into double standard curves to obtain the initial template copy numbers of the GAPDH gene and the hlyz gene in the genome DNA sample. The GAPDH gene is present in a single copy in the genome of pichia pastoris, so the lysozyme gene copy number is equal to the starting template copy number of the human lysozyme gene/GDPDH gene starting template copy number.

The analysis result shows that the genome of the recombinant strain HC243 contains 5 genes of human lysozyme.

Example 3 construction of multicopy recombinant plasmid pPICZ α -hlyzV of human lysozyme

Based on pPICZ α A plasmid, 5 steps of assembly are carried out to construct recombinant plasmid pPICZ α -hlyzV containing human lysozyme gene reading frames with 5 copy numbers, as shown in figure 2, and the specific operation method is as follows:

① cloning hlyz-TT between EcoRI and BsmBI of pPICZ α A to obtain recombinant plasmid pPICZ α -hlyzTT, which is specifically performed by firstly extracting pPICZ α A plasmid, performing double digestion by EcoRI and BsmBI, recovering and purifying gel, performing PCR amplification by using high-efficiency Fidelity enzyme PhaxSupper-delFidelity DNA Polymerase of Nanjing Nozaki Biotech limited company (Vazyme Biotech Co., Ltd.) by using primer hlyz TT-1-F: gagaaaagagaggctgaagctgaattcaaggtctttgagagatgcga and hlzTT-1-R: CTCGAGGTACCGATCCGAGACGACTTCTCACTTAATCTTCTGTAC by using plasmid pPIC9k-hlyz as DNA template, obtaining hlyz-TT fragment, recovering and purifying PCR product by gel, performing ligation reaction of purified PICZ α A digested product and PCR product of yz-TT by using one-step cloning kit, transforming into E.zegli 5 α, culturing competent cells by PCR product containing low-level cDNA, and verifying resistance by using low-level cloning LB- α. mu.g.

② cloning hlyzORF between BsmBI and KpnI of pPICZ α -hlyzTT to obtain recombinant plasmid pPICZ α -hlyzORFII with 2 copies of hlyz reading frame, specifically, cloning pPICZ α -hlyzTT plasmid obtained in step ① by BsmBI and KpnI, and purifying and recovering by using gel recovery Kit, PCR amplifying by using primers hlyzORF-1-F: gaagattaagtgagaagtcgtctcagatctaacatccaaagac and hlyzORF-1-R: GCCGCCGCGGCTCGAGGTACCTCTCACTTAATCTTCTGTAC and pPIC9k-hlyz as DNA template, using high fidelity ORFII fragment by PCR amplification using high fidelity, recovering and purifying PCR product by gel, cloning Kit One step of Nanjing PyzORFII, cloning pPICZ digested product and PCR product of 82923. mu. hocht, transforming PCR product into plasmid containing low-content plasmid through PCR amplification reaction, PCR amplification by using plasmid containing plasmid DNA, plasmid DNA.

③ cloning hlyzORF between KpnI and SacII of pPICZ α -hlyzORFII to obtain 3 copies of recombinant plasmid pPICZ α -hlyzORFIII, which is prepared by digesting pPICZ α -hlyzORFII plasmid obtained in step ② with KpnI and SacII and recovering the plasmid through gel recovery kit, PCR amplifying with primer hlyzORF-2-F: cagaagattaagtgagaggtaccagatctaacatccaaagacgaaag and hlyzORF-2-R: GAAAGCTGGCGGCCGCCGCGGTCTCACTTAATCTTCTGTAC and pPIC9k-hlyz as DNA template to obtain high fidelity fragment of FIyzORFIII, recovering and purifying PCR product, and ligating the PCR product of pPICZ α -hlyzORFII digested product and PCR product of hlyzORFIII through one-step cloning kit, converting into E.colzei DH 23, sequencing with high fidelity cell, culturing with low-level pPICZ 82925 g/mL recombinant plasmid pPICyzORFIII, and verifying the resistance.

④ cloning hlyzORF between SacII and XbaI of pPICZ α -hlyzORFIII to obtain recombinant plasmid pPICZ α -hlyzORFIV with 4 copies of hlyz reading frame, which is prepared by digesting pPICZ α -hlyzORFIII plasmid obtained in step ③ with SacII and XbaI and purifying and recovering with gel recovery kit, PCR amplifying with primers hlyzORF-3-F: gagtacagaagattaagtgagaccgcggagatctaacatccaaagacgaaag and hlyzORF-3-R: CTTCTGAGATGAGTTTTTGTTCTAGATCTCACTTAATCTTCTGTAC with pPIC9k-hlyz as DNA template and using high fidelity enzyme to obtain fragment of hlzORFIV, gel recovering and purifying PCR product, ligating the purified pPICZ α -hlyzORFIII digested product with PCR product of hlyzORFIV with one-step cloning kit, and transforming into E.colzei DH 23, sequencing with high fidelity cell, culturing with low fidelity pPICZ 829 25 g/mL, and screening for low fidelity gene expression of recombinant plasmid pPICZ 4-hlyzORFIV.

⑤ fusion of histidine dehydrogenase gene HIS4 fragment with hlyzORF, cloning into recombinant plasmid pPICZ α -hlyzV with 5 copies of hlyz reading frame in step ④, specifically, cloning pPICZ α -hlyzORFIV plasmid obtained in step ④ between XbaI and NcoI, carrying out double digestion with XbaI and NcoI, and purifying and recovering with gel recovery kit, using primers His-F gtacagaagattaagtgagatctagatggagatgcaaaaacaatttc and His-R CTCCAGAAGGTTCTTATACCAAAC, using pPIC9k-hlyz as DNA template, carrying out PCR amplification with high fidelity enzyme to obtain HIS 9 fragment, recovering and purifying PCR product with gel recovery, using primers P-hlyz-F gtttggtataagaaccttctggagagatctaacatccaaagacgaaag and P-hlyz-R TTAAACACCACAACCTTGAACG, using pPIC9 6353-yz as DNA template, using high fidelity sequencing enzyme to obtain PCR amplification product, using primers P-hlyz-F6 4, purifying with high fidelity PCR amplification kit, using primers NcoHichlz DNA fusion DNA polymerase, cloning with DNA polymerase chain extension kit DNA fusion DNA, cloning with DNA fusion DNA polymerase, cloning with DNA fusion kit, cloning with DNA fusion kit, cloning DNA fusion, cloning with DNA fusion kit, cloning DNA fusion kit, purification with DNA fusion kit, purification with DNA fusion kit, purification of DNA fusion kit, purification with high fidelity, DNA recovery of DNA fusion kit, purification with high fidelity, DNA recovery of DNA fusion kit, purification with high fidelity kit, purification with DNA recovery kit, purification with high fidelity kit, purification with DNA recovery of DNA recovery kit, purification with high fidelity kit, purification.

Example 4 construction of a recombinant Pichia Strain IEF-hlyz15 containing multiple copies of the human lysozyme Gene

After the linearized pPICZ α -hlyzV plasmid constructed in example 3 is transformed into the recombinant Pichia pastoris strain HC243 obtained in example 2, resistant strains are screened by a Zeocin resistance plate, then strains with high-yield human lysozyme are screened by a bacteriolysis ring plate, and finally, a recombinant Pichia pastoris strain secreting and expressing human lysozyme is obtained by a triangular flask shaking experiment.

The specific operation is as follows:

① linearization of pPICZ α -hlyzV plasmid, inoculating E.coli DH5 α (pPICZ α -hlyzV) of the strain in example 3 to LB medium containing 100. mu.g/mL ampicillin, culturing overnight at 37 ℃, extracting pPICZ α -hlyzV plasmid by using a plasmid extraction kit, digesting the plasmid pPICZ α -hlyzV by using restriction enzyme SalI, linearizing the plasmid, recovering the digested product by using glue, and dissolving the purified linearized plasmid in ultrapure water;

② competent cells of the recombinant strain HC243 were prepared according to the method described in example 2.

③ the linearized pPICZ α -hlyzV is transformed into the recombinant strain HC243, positive transformants are selected, 80 mu L of the recombinant strain HC243 competent cells obtained in the step ② and 10 mu g of the plasmid pPICZ α -hlyzV linearized in the step ① are added into a 1.5mL precooling centrifuge tube and mixed evenly, then the mixed solution is transferred into a transformation cup (0.2cm type) pre-iced with ice, the transformation cup filled with the transformed mixed solution is iced for 5min, a Bio-Rad electric shock instrument is used for carrying out electric transformation, the voltage is 1.5kV, the electric field strength is 7.5kV/cm, the capacitance is 25 mu F, and the resistance is 400 omega, 1mL of 1M sorbitol aqueous solution in ice bath is added into the electric transformation cup immediately after pulse, then the transformed into a new 1.5mL centrifuge tube, standing culture is carried out for 2h at 30 ℃, 200 mu L of cell suspension is coated on a Zeocin resistance YPD plate containing 100 mu g/mL, and culture is carried out at 30 ℃ until colonies appear.

④ screening recombinant strains of human lysozyme by a lysis ring plate method, inoculating Micrococcus murauliticus ATCC4698 in 50mL of LB liquid culture medium, culturing at 37 ℃ for 18h, centrifuging at 3000 Xg for 10min, discarding supernatant, collecting cells, resuspending the cells in 5mL of sterile water, adding to 100mL of BMMY solid culture medium heated and boiled, mixing Micrococcus cells with the culture medium to obtain a bacterial carrying plate, spotting the bacterial colony growing on the Zeocin resistant plate obtained in the step ③ on the bacterial carrying plate, culturing at 30 ℃ for 3 days, continuously observing the change of the size of the lysis ring, and selecting the strain with larger lysis ring for the next round of screening.

⑤ according to the method of example 2, a flask shake flask fermentation was performed to screen a strain for high-yielding human lysozyme.

⑥ copy number detection of the human lysozyme gene of the high-activity recombinant strain was carried out according to the method in example 2.

Through the research, the recombinant pichia pastoris strain IEF-hlyz15 with high activity and copy number is obtained, and the copy number of the human lysozyme gene is 15.

Example 5 fermentation preparation of lysozyme by Strain IEF-hlyz15

First, culture of fermentation seed liquid

The recombinant Pichia pastoris strain IEF-hlyz15 of example 4 was streaked from the preserved Glycine max tube onto a YPD plate and cultured at 30 ℃ for 40 h; single colonies were picked, inoculated into YPD medium, and cultured at 30 ℃ and 200rpm for 20 hours to give a fermentation seed solution.

Two, 5L jar high density fermentation production lysozyme

The fermentation process of the 5L fermenter was divided into two stages: the growth stage of the thallus and the induced expression lysozyme stage. In the stage of thallus growth, the seed liquid is inoculated to a fermentation culture medium, an initial carbon source is utilized for growth and propagation, and when the carbon source is completely consumed, a certain amount of fresh carbon source (glycerol) is added, so that the cell density of the pichia pastoris is further improved. Starving and culturing for 1-1.5 hr without adding any substance when glycerol is supplemented and there is no residual glycerol in the fermenter, and adding methanolDuring the methanol induction culture stage, the methanol feeding rate is adjusted along with the induction stage and the dissolved oxygen in the tank. Sampling at different time points throughout the experimental procedure to determine OD₆₀₀And detecting lysozyme activity.

The specific flow of the fermentation tank experiment is as follows:

(1) glycerol batch fermentation stage: the volume of the initial fermentation medium was 2.5L and the volume of the fermentor was 5.0L. After the sterilization is finished, the tank body is correctly connected with a control device, and proper temperature parameters, stirring speed and ventilation volume (the temperature is 30 ℃, the stirring speed is 300rpm, and the ventilation volume is 5 m) are set³In/min). The pH of the fermentation broth was adjusted to 5.0 using ammonia and 10.875ml of a trace salt solution of PTM1 was added to the medium. After all parameters are stabilized, 100mL of seed solution which is freshly cultured for 20h is inoculated into the culture medium, and the volume inoculation amount is 4%. Adjusting the oxygen-dissolved electrode parameter to 100% before the fermentation stage, increasing the oxygen consumption rate of yeast along with the fermentation stage, and coupling stirring speed and Dissolved Oxygen (DO) to make DO value>20 percent. The batch fermentation phase lasts for about 20 hours, and then the glycerol is depleted, which is mainly characterized by a rapid rise in dissolved oxygen.

(2) And (3) glycerol feeding and fermenting stage: after the glycerol in the initial fermentation medium in the previous stage is completely consumed, the glycerol feeding fermentation stage is carried out, and the fermentation conditions are the same as the step (1). The glycerol feeding follows the principle of slow first and fast second, the feeding rate is 18mL/h/L of initial fermentation broth volume, 450mL of glycerol feeding culture medium is completely fed within 4 hours, and the dissolved oxygen rises again when the end mark of the glycerol feeding stage is finished. The key point is that the dissolved oxygen is controlled to be more than 20 percent, and the starvation culture is continued for 1 hour after the glycerol is supplemented.

(3) And (3) a methanol feeding fermentation stage: and after the glycerol feeding fermentation stage is completed, entering a methanol induction fermentation stage. The methanol addition process at this stage requires care and the methanol addition rate needs to be correlated with the growth of the biomass. If methanol is supplemented more quickly, too much methanol may result in the failure of the yeast cells to grow normally and even kill the cells. Once the cells begin to adapt to methanol, the oxygen demand is high and dissolved oxygen control becomes a critical loop. The methanol feed medium used for feeding was purified methanol supplemented with 12ml/LPTM 1. And (3) maintaining the initial methanol supplement rate at 3.6mL/hr/L of the initial fermentation culture medium, maintaining the dissolved oxygen DO to be more than 10%, fermenting for 2h, increasing the methanol supplement rate to 6.0mL/h/L of the initial fermentation culture medium, after fermenting for 3h, continuously increasing the methanol supplement rate to 9.0mL/h/L of the initial fermentation culture medium, and continuously fermenting for 115 h.

And (3) finishing the high-density fermentation process after the methanol feeding stage is finished to obtain fermentation liquor containing lysozyme, wherein the whole fermentation process approximately lasts for about 125 hours, the parameter change and the enzyme activity generation condition of the fermentation process are shown in fig. 7 and 8, the final fermentation OD value is 380, and the enzyme activity is 220 kU/mL.

Sequence listing

<110> Zhejiang New Yinxing Biotechnology Limited, Zhejiang Industrial university

<120> recombinant pichia pastoris engineering bacteria containing high copy number human lysozyme gene and application thereof

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>393

<212>DNA

<213> Unknown (Unknown)

<400>1

aaggtctttg agagatgcga attggctaga actttgaaga gattgggtat ggatggttac 60

agaggtattt ctttggctaa ctggatgtgt ttggctaagt gggaatctgg ttacaacact 120

agagctacta actacaacgc tggtgataga tctactgact acggcatctt ccaaattaac 180

tcccgttact ggtgtaacga tggtaagact ccaggtgctg ttaacgcttg tcatttgtct 240

tgttctgctt tgttgcagga taacattgct gatgctgttg cttgtgctaa gagagttgtt 300

agagatccac aaggtattag agcttgggtt gcttggagaa acagatgtca aaacagagat 360

gtccgtcaat acgttcaagg ttgtggtgtt taa 393

<210>2

<211>130

<212>PRT

<213> Unknown (Unknown)

<400>2

Lys Val Phe Glu Arg Cys Glu Leu Ala Arg Thr Leu Lys Arg Leu Gly

1 5 10 15

Met Asp Gly Tyr Arg Gly Ile Ser Leu Ala Asn Trp Met Cys Leu Ala

20 25 30

Lys Trp Glu Ser Gly Tyr Asn Thr Arg Ala Thr Asn Tyr Asn Ala Gly

35 40 45

Asp Arg Ser Thr Asp Tyr Gly Ile Phe Gln Ile Asn Ser Arg Tyr Trp

50 55 60

Cys Asn Asp Gly Lys Thr Pro Gly Ala Val Asn Ala Cys His Leu Ser

65 70 75 80

Cys SerAla Leu Leu Gln Asp Asn Ile Ala Asp Ala Val Ala Cys Ala

85 90 95

Lys Arg Val Val Arg Asp Pro Gln Gly Ile Arg Ala Trp Val Ala Trp

100 105 110

Arg Asn Arg Cys Gln Asn Arg Asp Val Arg Gln Tyr Val Gln Gly Cys

115 120 125

Gly Val

130

Claims

1. A recombinant pichia pastoris engineering bacterium containing a high-copy-number humanized lysozyme gene is characterized in that the recombinant pichia pastoris engineering bacterium is obtained by integrating a lysozyme gene shown as SEQ ID NO.1 into a pichia pastoris genome at a high copy number.

2. The recombinant Pichia pastoris engineering bacteria of claim 1, wherein the Pichia pastoris is Pichia pastoris (Pichia pastoris) GS 115.

3. The recombinant pichia pastoris engineered bacterium of claim 1, wherein the high copy number is 15 for an egg white lysozyme gene.

4. The recombinant pichia pastoris engineering bacteria of claim 1, wherein the amino acid sequence of the human lysozyme gene coding protein is SEQ ID No. 2.

5. The recombinant pichia pastoris engineering bacteria of claim 1, wherein the construction method of the recombinant pichia pastoris engineering bacteria comprises the following steps:

1) cloning the human lysozyme gene shown in SEQ ID NO.1 to a secretory expression plasmid pPIC9K to obtain a recombinant plasmid which is marked as a recombinant plasmid pPIC 9K-hlyz;

2) after linearization is carried out on the recombinant plasmid pPIC9K-hlyz by using restriction enzyme SacI, the linearized plasmid is converted into a pichia pastoris strain, and a high-activity recombinant strain with the copy number of 5 is obtained by screening and is marked as a recombinant strain HC 243;

3) cloning a human lysozyme gene expression reading frame on the recombinant plasmid pPIC9K-hlyz to a pPICZ α A plasmid, and constructing a recombinant expression plasmid with 5 copy numbers, wherein the recombinant expression plasmid is marked as a recombinant expression plasmid pPICZ α -hlyzV;

4) after the recombinant expression plasmid pPICZ α -hlyzV is linearized by a restriction enzyme SalI, a recombinant strain HC243 is transformed, and a high-activity recombinant strain with the copy number of 15 is screened and recorded as a recombinant strain IEF-hlyz 15.

6. An application of the recombinant pichia pastoris engineering bacteria of claim 1 in preparing lysozyme by fermentation.

7. Use according to claim 6, characterized in that the fermentation process is as follows: (1) streaking the recombinant Pichia pastoris engineering bacteria on a YPD flat plate, and culturing for 40h at 25-30 ℃; inoculating the single colony into a seed culture medium, culturing at 25-30 ℃ and 250rpm under 100-; the YPD plates consisted of: 10g/L of yeast powder, 20g/L of peptone, 10g/L of glucose and 15g/L of agar powder, wherein the solvent is deionized water, and the pH value is natural; the final concentration composition of the seed culture medium is as follows: 10g/L of yeast powder, 20g/L of peptone and 10g/L of glucose, wherein the solvent is deionized water and the pH value is 7.0;

2) fermentation culture: inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 1-10% of the volume of the seed liquid to perform glycerol batch fermentation at the temperature of 25-30 ℃ and the dissolved oxygen DO of more than 20%, and fermenting for 18-24h until the glycerol is exhausted; starting to enter a glycerol feeding fermentation stage: the supplementing rate of the glycerol supplementing culture medium is 18-20mL/h/L of initial fermentation broth volume, the glycerol supplementing culture medium is fermented for 4-6h under the conditions that the temperature is 25-30 ℃, the stirring rate is 200-900 rpm, and the ventilation volume is 2-4L/min/L of initial fermentation culture medium, the stirring rotation speed is controlled in the fermentation process to be coupled with the dissolved oxygen DO value, the DO value is controlled to be more than 20%, and starvation culture is continued for 1-2h after the glycerol is exhausted again; entering a methanol induction fermentation stage, controlling the feed rate of a methanol feed medium to be 3.0-3.6mL/h/L of an initial fermentation medium, maintaining the dissolved oxygen DO to be more than 10%, fermenting for 2-3h, increasing the feed rate of the methanol feed medium to be 5.0-7.2mL/h/L of the initial fermentation medium, after fermenting for 3-5h, continuously increasing the feed rate of the methanol feed medium to be 9.0-10.2mL/h/L of the initial fermentation medium, continuously fermenting for 110-120h, and finishing fermentation to obtain fermentation liquor containing lysozyme; the fermentation medium comprises the following components in final mass concentration: 26.7 ml/L85% phosphoric acid, 0.93g/L calcium sulfate, 18.2g/L potassium sulfate, 14.9g/L MgSO₄·7H₂O, 4.13g/L of potassium hydroxide, 40g/L of glycerol, 4.35ml/L of PTM1 buffer solution, deionized water as a solvent and pH5.0; the glycerol feed medium is prepared by adding 12ml/L PTM1 buffer solution into 25% glycerol by mass concentration; the methanol feeding medium is prepared by adding 12ml/L PTM1 buffer solution into anhydrous methanol; the PTM1 buffer composition: 6.0g/L CuSO₄·5H₂O, 0.08g/L sodium iodide, 3.0g/L MnSO₄·H₂O，0.2g/L Na₂MoO₄·2H₂O, 0.02g/L boric acid, 0.5g/L cobalt chloride, 20.0g/L zinc chloride and 65.0g/L FeSO₄·7H₂O, 0.2g/L biotin, 5.0ml/L sulfuric acid and deionized water as a solvent.

8. Use according to claim 7, characterized in that the glycerol feed medium is supplemented in an amount of 15-20% of the volume of the fermentation medium originally added.

9. The use according to claim 7, wherein the methanol feed medium is supplemented in an amount of 50-80% of the volume of the fermentation medium as originally added.